17188
J. Phys. Chem. B 2004, 108, 17188-17197
Interactions of a Sulfonated Aluminum Phthalocyanine and Cytochrome c in Micellar Systems: Binding and Electron-Transfer Kinetics Ce´ sar A. T. Laia* and Sı´lvia M. B. Costa Centro de Quı´mica-Estrutural, Complexo 1, Instituto Superior Te´ cnico, 1049-001 Lisboa, Portugal ReceiVed: June 2, 2004; In Final Form: August 23, 2004
The interaction of the tetrasulfonated aluminum phthalocyanine with cytochrome c was studied in micellar systems, after previous work that was conducted in aqueous solutions [Laia et al., J. Phys. Chem. B, 2004, 108, 7506-7514]. It is found that, in ionic micellar aggregates, the interaction is disrupted whereas, in neutral micelles and reversed micelles, it is maintained. The binding constant decreases, but it still reaches high values in neutral micellar systems. The electrostatic nature of the complex formation was assessed in sucrose/ water mixtures and was observed to be strongly dependent on the dielectric constant of the mixture. The electron-transfer process within the complex is affected by the microviscosity, leading to faster decays in more-viscous systems, which is attributed to small dynamic conformational changes of the complex. In Triton X-100 micelles, the result is similar to that obtained in aqueous solution; however, in Brij 35 micelles, it shows features that are unique, reflecting an apolar environment. High electron-transfer rate constants are obtained in cyclohexane/n-hexanol/Triton X-100/water microemulsions, and these rate constants cannot be atributed solely to a more-viscous environment. It is suggested that, in such systems, other effects, such as confinement and water structure, have a major role.
1. Introduction Electron-transfer reactions are ubiquitous in nature; they have a key role in photosynthesis and respiration.1-7 Those processes occur in membranes that provide a microheterogeneous environment that maximizes the electron transfer efficiency through self-assembly of lipids and proteins in a correct manner.8-13 The understanding of these processes may provide important knowledge about drug design or solar energy storage.1-14 The use of mimicking architectures of membranes such as vesicles, sol-gel systems, or micelles may also provide a deep understanding of how the processes occur in nature.8-14 The involvement of proteins in such reactions presents an additional challenge, because of the fact that the environment affects its overall structure and the interaction with solutes is either enhanced or disrupted.8,9,11-13,15-31 The proteins and complex media such as membranes or micelles also present slow relaxation patterns, which can slow the dynamics of chemical reactions in such systems.3,10,32-42 On the other hand, confinement effects can increase the local concentration of the solutes, thus producing a catalytic effect, or the low polarity can increase the rate of several chemical reactions. Cytochrome c (Cyt c) is not a very large protein; it has a heme group resting in the globule.1,4,15-23,25-29,43-59 It is watersoluble, with a high positive charge (approximately +8) at neutral pH. The active site that allows the protein to dock on other proteins such as Cytochrome c Oxidase (Cyt c Ox)15,19,23,28,44,45,48 or caspase proteins,60 which have important roles in respiration and apoptosis, has high positive charge density. This allows a strong binding to solutes with a high negative charge that mimic the architecture of the active site of Cyt c Ox and caspases.7,8,16-20,24-26,28-31 Anionic porphyrins and phthalocyanines, * Author to whom correspondence should be addressed. Telephone: +351 218419274. Fax: +351 218464455. E-mail address: Cesar.laia@ popsrv.ist.utl.pt.
as well as calixarenes and dendrimers designed to interact with Cyt c, proved that such molecules could bind with equilibrium constants as high as those found in nature (>105 M-1). The interaction is mainly driven by electrostatic interaction45 (although some contribution of hydrophobic interactions is also important) and dissipates as the ionic strength increases. On the other hand, the effect on the binding of dyes in organized media such as membranes is still largely unexplored. Previous work has shown that electron-transfer from organic donors to the Cyt c heme group proceeds at rates of ∼108 s-1,1,4,41-43,46,47 depending on the free energy of the reaction and the electronic coupling between the donor and acceptor. Recently, rate constants as high as 109 s-1 were reported for reactions involving phthalocyanines and fullerene dendrimers.31 In self-assemblies such as membranes or micelles, however, the data is scarce; thus, little is known about the magnitude of the electron-transfer rates involving Cyt c complexes. The interaction of tetrasulfonated aluminum phthalocyanine (AlPcS4) with Cyt c in water was studied recently.61 It was shown that a strong binding occurs with an equilibrium constant of ∼3 × 105 M-1. Electron transfer occurs from the AlPcS4 singlet excited state to the Cyt c with a high rate, when compared to similar systems (∼109 s-1). This indicates a strong electronic coupling between the donor and acceptor, presumably due to the fact that the distance between them is comparatively small. Thus, the binding occurs in a region of the Cyt c where electron transfer is more efficient. Cyt c in microheterogeneous environments such as micelles62 or polymers63 undergoes structural changes that have an impact on the heme group. Such changes are likely to happen in the reversed micelles also, although the protein activity remains unchanged. In fact, Bertini et al. showed that a molten globule protein with altered axial ligation is formed under such conditions.62 The present work reports the behavior of a AlPcS4/
10.1021/jp047616l CCC: $27.50 © 2004 American Chemical Society Published on Web 10/13/2004
Cytochrome c Complexes in Micellar Systems Cyt c complex in organized media (micelles and reversed micelles). It is shown that ionic surfactants dissociate the complex, probably because there is a competition between the phthalocyanine and the surfactant molecules for the Cyt c active site. However, in neutral micelles, the binding constant is the same order of magnitude as that observed in water. The fluorescence quenching due to electron transfer from the AlPcS4 singlet excited state to the heme group proceeds with a high rate constant (>109 s-1) and the kinetics is nonexponential. Interestingly, significant differences are observed between Triton X-100 micelles and Brij 35 micelles, which are likely to be related to the structure of the molecular aggregates.64-69 In Triton X-100 microemulsions, the structure is strongly dependent on the water content38,70-72 and, thus, has a marked effect on the reaction kinetics, leading to results that are similar to those found either in Triton X-100 micelles or Brij 35 micelles. The reasons for this behavior are discussed. 2. Experimental Section AlPcS4 was purchased from Porphyrin Products (99% purity) and used as received. The phthalocyanine reagent is a mixture of three regioisomers. Previous work with disulfonated phthalocyanines showed that the photophysical properties remain unchanged in all isomers.73 Cyt c was purchased from Aldrich (97% purity) and used without further purification. Solvents used were bidistilled water and spectroscopic cyclohexane and n-hexanol from Aldrich. Cetyl trimethylammonium bromide (CTAB), Triton X-100, and Brij 35 surfactants were purchased from Sigma and used as received. The Triton X-100 reversed micelles were prepared by adding n-hexanol and Triton X-100 in cyclohexane to have a solution with a Triton X-100 concentration of [Triton X-100] ) 0.2 M and a 3:2 (w/v) ratio of Triton X-100 to n-hexanol. Water was added afterward to obtain the required water content, w0 (w0 ) [H2O]/[Triton X-100]). Absorption spectra were recorded at room temperature with a Jasco model V-560 UV/VIS absorption spectrophotometer. Steady-state emission measurements were recorded with a Perkin-Elmer LS 50B spectrofluorimeter with the sample holder thermostated at 22 °C. The instrument response at each wavelength was corrected by means of a curve provided with the instrument. Time-correlated single photon counting was used to obtain the fluorescence decays in a Picoquant model MicroTime 200 fluorescence lifetime microscope system. The excitation source consisted of a pulsed red-diode laser (PDL 800, PicoQuant Berlin) with a wavelength of λ ) 635 nm, providing output pulses of
